One of the central problems in visual science is the mechanism of spectral tuning among the visual pigments. How is it that the absorption maxima can range from 380 nm to over 600 nm when the actual molecule that absorbs light, the 11-cis-retinal chromophore, is identical in all of the pigments? Clearly, there are interactions of the retinal chromophore with amino acid sidechains in the proteins that modify the spectral properties the chromophore. It is the purpose these studies to unravel these mechanisms for one extremely well defined experimental system: the human color vision pigments. This work will focus on three groups of amino acids in these proteins known to profoundly affect the spectral properties of the chromophore: 1) the 7 amino acids that cause the spectral difference between the red and green pigments; 2) the protonated Schiff base of the chromophore and Schiff base counterion; and 3) the chloride binding site of the red and green pigments. The approach will be to use site-directed mutagenesis, chemical modification of the proteins, and chromophore analogs in combination with in vitro characterization of the purified pigments. As a result of a recent technical breakthrough in the application of resonance Raman spectroscopy to the study of these pigments, this powerful spectroscopic tool will be used extensively to probe the structure of the retinal chromophore in all aspects of this work. The enormous potential of this application is apparent from a realization that the source of visual pigment for the Raman studies will be a library of over 150 mutants in the blue, green and red visual pigments that was constructed over the original grant period.